Approximately 50% of patients presenting with breast cancer have lymph node-negative tumors. Although the overall prognosis for such patients is substantially better than for those with positive node. 30% will still succumb to metastatic disease. Experience with node-positive disease suggests that this false-negative patient cohort may derive significant survival benefit form adjuvant systemic therapy. Expression of the c-erbB-2 (neu, HER-2) receptor has been proposed as one criterion for predicting the natural history of human breast cancer, but the predictivity of this receptor in early-stage disease remains uncertain. My previous work established that c-erbB-2 receptors exist in two interconvertible forms- an activated 175kDa(p(175) tyrosine- phosphorylated form, and an inactive 185kDa(p185) tyrosine- dephosphorylated form. This work led to the characterization of p175 expression in human tumor cells using activation-specific antibodies created by immunizing animals with synthetic phosphopeptides duplicating the sequence of a major c-erbB-2 autophosphorylation site. The present proposal aims to clarify the pathogenetic and prognostic significance of the c-erb-B-2 phenotype in human breast cancer by exploiting this novel technology. The project has three objectives: The first objective is to determine the signalling significance of individual c-ebb-2 receptor autophosphorylation sites by generating polyclonal antisera to the relevant phospho- and dephosphopeptides: The second objective is to raise activation- and inactivation-specific monoclonal antibodies to phosphopeptide sequences identifying different c-ebb-2 receptor activation states. The third objective is to use these activation-specific monoclonal antibodies to define expression of p175 and p185 in pre-invasive, invasive and metastatic breast tumors, and to correlate presence of these receptor isoforms with expression of the c-ebb-2 ligand (heregulin) at the mRNA and protein levels. These clinicopathologic correlations will establish the feasibility of a prospective randomized study of adjuvant systemic treatment based on expression of p175, p185 and heregulin in node-negative breast tumors. This goal is both achievable and timely given the advent of activation- state-specific phosphoprotein immunodetection (APHID) technology, the track record of the CRC Breast Group in the characterization of human breast tumors in vivo, and the recent amalgamation of Charing Cross Medical School with Imperial College of Science and Technology. The most important factor favoring success in this project, however, is that the CRC Laboratories provided unlimited access to a bank of 4000 frozen breast cancer specimens, together with computerized databases supplying all details of breast cancer clinical presentation, disease stage, therapeutic response and survival outcome.